Assembly of Multigene Constructs Using Golden Gate Cloning

  • Sylvestre Marillonnet
  • Stefan Werner
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)


Efficient DNA assembly methods are required for synthetic biology. Standardization of DNA parts is an essential element that not only facilitates reuse of the same parts for various constructs but also allows standardization of the assembly strategy. We provide here a protocol for assembly of multigene constructs from standard biological parts using the modular cloning system MoClo. Making constructs using this system requires to first define the structure of the final construct and to identify all basic parts and vectors required for the construction strategy. The cloning strategy is in large part determined by the structure of the final construct, which is then made using a series of one-pot Golden Gate cloning reactions.

Key words

Modular cloning Synthetic biology Biological parts DNA assembly Multigene constructs 


  1. 1.
    Schneider JD, Marillonnet S, Castilho A et al (2014) Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana. Plant Biotechnol J. doi: 10.1111/pbi.12184 PubMedCentralPubMedGoogle Scholar
  2. 2.
    Castilho A, Strasser R, Stadlmann J et al (2010) In planta protein sialylation through overexpression of the respective mammalian pathway. J Biol Chem 285:15923–15930PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Knight T (2003) Idempotent vector design for standard assembly of Biobricks. MIT Artificial Intelligence Laboratory; MIT Synthetic Biology Working Group. Accessed 27 June 2014
  4. 4.
    Weber E, Engler C, Gruetzner R et al (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6, e16765PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e3647PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Engler C, Youles M, Gruetzner R et al (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol. doi: 10.1021/sb4001504 PubMedGoogle Scholar
  7. 7.
    Engler C, Marillonnet S (2013) Combinatorial DNA assembly using Golden Gate cloning. Methods Mol Biol 1073:141–156PubMedGoogle Scholar
  8. 8.
    Engler C, Marillonnet S (2014) Golden Gate cloning. Methods Mol Biol 1116:119–131PubMedGoogle Scholar
  9. 9.
    Werner S, Engler C, Weber E et al (2012) Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioengineered Bugs 3:38–43PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sylvestre Marillonnet
    • 1
  • Stefan Werner
    • 2
  1. 1.Department of Cell and Metabolic BiologyLeibniz-Institut für PflanzenbiochemieHalleGermany
  2. 2.Nomad Bioscience GmbHHalleGermany

Personalised recommendations